Project Summary Tissue stiffness varies along organs and tissue types, from very soft (e.g. brain) to intermediate (e.g. digestive organs) and to very stiff (e.g. bone). Sensing this stiffness by cells determines differentiation, proliferation, migration and survival, which are all important for development. Local tissue stiffening is a hallmark of cancer, sensing of which by cancer cells causes further tumor progression and metastasis. Understanding stiffness sensing mechanism is thus essential for designing appropriate treatment strategy against developmental disorders and cancer. The prominent ?sensor? for mechanical stiffness of tissue environment is a molecular complex located between the cell and the environment, referred to as a focal adhesion. The first step in stiffness sensing is thought to involve transmission of increased level of a force across molecules in the focal adhesion against higher tissue stiffness, but the underlying biomechanical/molecular mechanisms remain poorly understood. The challenge in investigating this mechanism is in the dynamic nature of its structure, molecular interaction and mechanical forces affecting its assembly. The goal of this work is to understand whether, how and when the focal adhesion during dynamic assembly from their birth starts to be able to transmit the differential forces against varying tissue stiffness levels. Toward this goal, we have developed a set of experimental, microscopic and computational techniques to measure a small force from the adhesions, to profile heterogeneous dynamics of entire adhesion population, and to link molecular activity to force transmission in statistically confident manner. Specifically, we focus on a tiny dot-like complex called ?a nascent adhesion?, an early form of the focal adhesion, and involvement of the mechanical linker protein talin, given its ability to be stretched and expose binding sites for other molecules like a vinculin, another mechanical linker protein. The overall objective of this proposal is to use these techniques to test a novel conceptual model of stiffness sensing in which nascent adhesions are sensitive to the tissue stiffness by talin-mediated transmission of force, which promotes further maturation of them to focal adhesions. We will determine if 1) nascent adhesions can transmits differential levels of force in response to varying ECM stiffness, 2) the main source of force actin cytoskeleton- driven and myosin-independent, 3) there is concurrent recruitment of talin and vinculin as a pre-assembled complex and whether it affects force-transmission in response to the ECM stiffness, and 4) competition for talin binding of talin-binding proteins against actin-binding decreases force transmission by nascent adhesion and impair the ECM stiffness sensing. An enhanced mechanistic understanding of these processes would increase our fundamental knowledge of how cells sense and respond to tissue mechanics. Thus, the proposed studies are relevant to the NIH's mission, as they will lead to new insights in physiology and pathophysiology including tissue development, regeneration and cancer progression.